Sequence of chondrocranial development in basal anurans—Let’s make a cranium

Background The craniofacial skeleton is an evolutionary innovation of vertebrates. Due to its complexity and importance to protect the brain and aid in essential functions (e.g., feeding), its development requires a precisely tuned sequence of chondrification and/or ossification events. The comparison of sequential patterns of cartilage formation bears important insights into the evolution of development. Discoglossus scovazzi is a basal anuran species. The comparison of its chondrocranium (cartilaginous neuro- & viscerocranium) development with other basal anurans (Xenopus laevis, Bombina orientalis) will help establishing the ancestral pattern of chondrification sequences in anurans and will serve as basis for further studies to reconstruct ancestral conditions in amphibians, tetrapods, and vertebrates. Furthermore, evolutionary patterns in anurans can be studied in the light of adaptations once the ancestral sequence is established. Results We present a comprehensive overview on the chondrocranium development of D. scovazzi. With clearing and staining, histology and 3D reconstructions we tracked the chondrification of 44 elements from the first mesenchymal Anlagen to the premetamorphic cartilaginous head skeleton and illustrate the sequential changes of the skull. We identified several anuran and discoglossoid traits of cartilage development. In D. scovazzi the mandibular, hyoid, and first branchial arch Anlagen develop first followed by stepwise addition of the branchial arches II, III, and IV. Nonetheless, there is no strict anterior to posterior chondrification pattern within the viscerocranium of D. scovazzi. Single hyoid arch elements chondrify after elements of the branchial arch and mandibular arch elements chondrify after elements of the branchial arch I. Conclusions In Osteichthyes, neurocranial elements develop in anterior to posterior direction. In the anurans investigated so far, as well as in D. scovazzi, the posterior parts of the neurocranium extend anteriorly, while the anterior parts of the neurocranium, extend posteriorly until both parts meet and fuse. Anuran cartilaginous development differs in at least two crucial traits from other gnathostomes which further supports the urgent need for more developmental investigations among this clade to understand the evolution of cartilage development in vertebrates.

(e.g., [3]). The jaws derive from mesenchyme of the first or mandibular arch, the hyoid elements derive from the 2nd or hyoid arch, and branchial (gill bearing) or posterior pharyngeal elements derive from arches 3 to 6 (in some vertebrates more arches are present) (e.g., [4]).
Amphibians are the most basal four-legged 'terrestrial' vertebrates (Tetrapoda) and comprise salamanders, frogs, and caecilians. Frogs are distributed worldwide except at the poles and Greenland, and inhabit a wide range of ecosystems [5,6]. Most frogs have a biphasic lifestyle; that is, they pass through a free-swimming aquatic larval (tadpole) stage which is connected via a remodeling process called metamorphosis to the often terrestrial adult stage (frog). Tadpoles have mostly cartilaginous cranial elements; hence their heads are mainly supported by chondrocranial elements. Therefore, the chondrocranium sets the baseline for further development of the head [7]. Bony elements do not occur until the onset of metamorphosis. The tadpoles are as diverse as the adults with tiny predatory, giant megalophagous, medium sized scraping or filter feeding tadpoles and many other varieties [6]. All the different feeding types are associated with adaptations in the cranial skeleton and musculature.
All differences in mature larvae have to be caused by differences during the development. Previously, Ziermann et al. [8] studied the sequence of development of head muscles in larvae of frogs and compared these between each other and with salamanders and the Australian lungfish. They did not find any correlation of developmental sequence and feeding style, size, or habitat. However, they found that cephalic muscles tend to develop from anterior to posterior with 1st and 2nd arch-derived muscles developing before the 3rd pharyngeal arch-derived muscles, and these before the 4th and 5th pharyngeal arch muscles. They also described that there is a pattern of the muscles developing and differentiating first externally (more superficial) before medially (deeper) located muscles. Lastly, muscles tend to develop from their origin towards their insertion [8]. Several other studies in other species confirmed the general trends (chick: [9]; lungfish: [10,11]; small spotted catshark: [12]); but although pointed out that the mandibular arch seems to develop more randomly than the other arches [12], which could support the hypothesis that the mandibular arch was secondarily added to the pharyngeal or branchial arches [13].
Numerous investigations on the chondrocranial development are available and numerous species from all major vertebrate clades were added to the account recently [14-23, 23, 24]. The early chondrocranial development is similar within birds and variation relates to developmental patterns rather than to the presence or absence of specific elements [24]. A posterior anterior direction of chondrification of the skull was described in birds and squamates [19,23,24] whereas at least the cartilaginous visceral skeleton of several vertebrates develops in anteroposterior direction [14,17,18,20]. Shared features of chondrocranial development as well as sequential differences are helpful for further analyzing phylogenetic relation and evolutionary pathways of cartilaginous development.
Although ossification sequences are available for numerous frog species, investigations on the sequence of larval cartilage development are scarce. The sequence of development for chondrocranium elements was recently studied for two basal frog species (Xenopus laevis and Bombina orientalis). These studies identified several patterns which may be candidates for the basal pattern of cartilage formation in anurans [21,25]. (1) The first mesenchymal Anlage which appears during the chondrocranial development is the Anlage of the ceratohyal, which is also the first structure that chondrifies. (2) Anterior to posterior sequences of chondrification are present in neurocranial structures which encapsulate the sensory organs and the brain, the neurocranium-anchoring processes of the palatoquadrate, and the four ceratobranchials that form the branchial basket.
In order to analyze if general developmental patterns can be identified or if there is a correlation with phylogeny, lifestyle, and/or size, we will here lay the groundwork for a larger scale comparative development study. We compare the sequence of chondrocranial development in three species Discoglossus scovazzi, Xenopus laevis, and Bombina orientalis (Fig. 1). X. laevis and B. orientalis were previously described [21,25] and we only updated their developmental sequences where needed. The chondrocranium development of D. scovazzi will be described in detail.
The present work provides a comprehensive overview on the cartilage development of D. scovazzi from the first mesenchymal Anlagen until the premetamorphic cartilaginous head skeleton. This overview completes recent and sets the standards for future investigations on the chondrocranial development of larval anurans. Furthermore, we propose a defined set of structures, abbreviations and a reliable workflow for the analysis of this development. We present baseline data which is intended to be used in future comparative analyses of the cartilaginous development in anurans as well as in other tetrapods. Such extensive analyses should shed light on the evolution of the cartilaginous development during ontogenesis. In the end we want to encourage scientists to not only rely on the description of single stages when describing tadpole morphology. A sequential description of the development, no matter if muscles, cartilages, nerves or other morphological structures are investigated, bears much more information and will help to further uncover the underlying pathways of the evolution of development. (Table 1)

Infrarostral cartilage
There is a single, V-shaped infrarostral cartilage in D. scovazzi. It is connected laterally to the paired Meckel's cartilage via an intramandibular commissure and the intramandibular joint. The first traces of the infrarostral appear at stage D2 ( Fig. 2) when the majority of cartilages are already present as mesenchymal Anlagen or differentiated chondroblasts (Fig. 3a). The mesenchymal Anlage of the infrarostral develops ventral to the anteroventral margin of the oral cavity and medially to the mesenchymal Anlagen of the Meckel´s cartilages (Fig. 2). The Anlage is already V-shaped. At stage D3 the intramandibular commissure becomes visible, which connects the dorsolateral part of the infrarostral and the ventromedial part of Meckel´s cartilage. The number of chondroblasts increase in the otherwise unchanged infrarostral so that in stage D4 only the median part still consists of dense mesenchymal cells. At the transition from stage D4 to C1 the chondroblasts differentiate into chondrocytes. In stage C2 the whole cartilage is surrounded by a distinct perichondrium. The medial parts of the infrarostral cartilage are connected by a medial synchondrosis. The small dorsolateral margin where the intramandibular joint develops chondrifies at stage C3.

Admandibular cartilage
The admandibular cartilages are small, vertically oriented rods between the suprarostral and the infrarostral cartilages. The rostrolateral tip of the admandibular cartilage is connected to the dorsomedian surface of Meckel´s cartilage through the small mandibulo-admandibular ligament. The mesenchymal Anlage is spherical and appears at stage D4 lateral to the infrarostral cartilage and ventromedial to Meckel´s cartilage (Fig. 2). The posterodorsal surface of the admandibular borders on the ventromedial surface of Meckel´s cartilage and does not interfere with the articulation between Meckel's cartilage and infrarostral cartilage. During further differentiation the admandibular Anlage extends ventrally and is rod-shaped when the first chondroblasts appear at stage C2 (Figs. 2 and 3a). The dorsal tip is in close proximity to the ventromedial process of Meckel´s cartilage. A differentiation into chondrocytes was not observed in the investigated stages.

Suprarostral cartilage
The plate-like suprarostral cartilage is the anteriormost cartilage. It supports the upper lip and consists of a median corpus and two lateral alae. Two dorsal projections of the corpus articulate with the anterior part of the trabecular horns. The alae emerge from the lateral margin of the corpus, bend posterolaterally and bear a short posterior process. The mesenchymal anlagen of the two alae and the corpus develop at stage A3 as two separated Anlagen which surround the anterior part of the oral cavity laterally and dorsally (Fig. 3a). Until stage A5 the mesenchymal Anlagen fuse medially and the Anlage acquires the typical plate-like shape of the suprarostral cartilage.

Meckel's cartilage
Meckel's cartilage is the ventral component of the mandibular arch and forms part of the lower jaw in many vertebrates. The lower jaw of many anuran larvae consists of two elements, the medial and more anterior infrarostral cartilage and the more lateral and more posterior located paired Meckel's cartilage. The latter articulates with the palatoquadrate via the mandibular joint. In D. scovazzi the Meckel's cartilage is slightly sigmoid and typically horizontally oriented. It consists of the clearly distinguishable retroarticular process dorsolaterally and an inconspicuous ventromedial process. The mesenchymal Anlagen of Meckel's cartilage and its processes are among the earliest visible skeletal structures during skeletogenesis. They arise at stage A2 as two distinct condensations beneath the ventrolateral margin of the oral cavity ( Fig. 3a).

Palatoquadrate
The palatoquadrate is the dorsal component of the mandibular arch. The jaw joint is formed by the articular process of the palatoquadrate and the retroarticular process of Meckel's cartilage. The articular process is the most anterior part of the palatoquadrate. The posterior parts, like the muscular process and the subocular bar, are the origin of numerous muscles which enable jaw and eye movement. The palatoquadrate borders the neurocranial structures laterally and is connected to them via the quadratocranial commissure anteriorly and the ascending process posteriorly. In several larval amphibians there is a third neurocranium anchoring structure, the larval otic process, which is missing in D. scovazzi. The mesenchymal Anlage of each palatoquadrate is bipartite and arises at stage A2 (Fig. 3b). The Anlage of the quadratocranial commissure is flanking the pharyngeal cavity laterally whereas the Anlagen of the articular process and the hyoquadrate process border the Anlage of the mandibular levator muscles ventrally. Until stage A3, the Anlage of the muscular process extends dorsolaterally, while the Anlage of the subocular bar extends posteriorly. The lateral Anlage of the quadratocranial commissure is now connected to the ventral Anlage of the palatoquadrate. The second neurocranial anchoring process, the ascending process, arises at stage A5 during further posterior extension of the Anlage of the palatoquadrate. The first parts of the palatoquadrate to differentiate into chondroblasts at stage D3 are the mesenchymal Anlagen of both neurocranium anchoring structures (Fig. 4). The muscular process extends further dorsally and is now flanked by the developing orbitohyoideus muscle laterally and mandibular levator muscles medially. Three processes (muscular, articular, hyoquadrate) and the subocular bar differentiate into chondroblasts at stage D4 ( Fig. 4). At the same stage the ligamentum tectum becomes visible as connective tissue between the dorsal tip of the muscular process and the neurocranium. While the Anlagen appear in a more or less antero-posterior pattern, the different parts of the palatoquadrate do not chondrify in anterior to posterior direction. Instead, the quadratocranial commissure and the subocular bar chondrify first at stage C1, whereas all other parts chondrify simultaneously at stage C2 (Fig. 4). The subocular bar becomes wider and flatter during further development. It extends posteriorly but never reaches the otic capsule.

Basihyal
The basihyal is a small, median cartilage anterior to the large ceratohyals. No muscles attach onto it. The mesenchymal Anlage develops at stage D4 between the anterior tips of the anterior processes of the ceratohyals (Fig. 3a).
The Anlage remains small during differentiation. At stage C2 the chondroblasts differentiate. At stage C4 the whole Anlage consists of chondrocytes which form a small, rounded plate-like cartilage.

Ceratohyal
The ceratohyal of D. scovazzi is a paired cartilage which each consists of a corpus, an anterior, a lateral, and a posterior process as well as an articular condyle. An anterolateral or postcondylar process is not present. The two separate mesenchymal Anlagen of the cartilage fuse via the median pars reuniens. The corpus, the lateral process, and the articular condyle are present as mesenchymal Anlagen at stage A2 (Fig. 3a, b). The corpus arises ventrally to the pharyngeal cavity. The Anlage of the orbitohyoideus muscle extends laterally to the lateral process.
The articular condyle is situated ventrally to the Anlage of the hyoquadrate process of the palatoquadrate. A gap between the two Anlagen closes at stage A3 when the mesenchymal Anlage of the pars reuniens, which connects both Anlagen anteriorly, arises. From stage A3 onwards only one Anlage of the ceratohyal is present. The mesenchymal Anlagen of the anterior and posterior processes also appear at this stage. The ceratohyal further widens laterally and the posterior process bulges posteriorly. At stage D1 the corpus is the first structure in which cells differentiate into chondroblasts. The articular condyle, and lateral and posterior processes are made of chondroblast by stage D4 (Fig. 5), followed by the anterior process at stage D5 and the pars reuniens at stage D6 (Fig. 3a). The lateral process becomes more pointed whereas the anterior process remains inconspicuous and flat. The pars reuniens extends posteriorly and is continuous with the basibranchial. All parts of the ceratohyal chondrify at stage C2 (Fig. 5), except the pars reuniens, which chondrifies at stage C3. The ceratohyal is horizontally oriented and the lateral parts bend posteriorly. The hyoquadrate process of the palatoquadrate and the articular condyle of the ceratohyal form a joint at stage C2.

Basibranchial
In D. scovazzi the basihyal is located medially between the ceratohyals. The pars reuniens connects the ceratohyal to the anterior part of the basihyal.   get closer to the hypobranchial plates while their lateral ends slightly bend dorsally. At stage D2 and D3 all remaining parts of the branchial basket became visible as mesenchymal Anlagen (Fig. 3a). Between the stages D4 (Fig. 6) and D6 all structures differentiate into chondroblasts. The ceratobranchials and the hypobranchial plate are still relatively thin, but they become thicker during the following stages. The chondroblasts of the hypobranchial plate and ceratobranchial I are the first to differentiate into chondrocytes at stage C1 followed by the remaining structures of the branchial basket at stage C2 (Fig. 6). The general shape of the branchial basket is acquired at this stage and the whole basket grows bigger during further development.

Neurocranium
The Neurocranium of D. scovazzi consists of a single ethmoid plate and bilaterally present a trabecular horn, an orbital cartilage, a marginal tectal taenia flanking the brain, a parachordal and an otic capsule which surrounds the inner ear. First traces of cartilaginous development are found within the neurocranium at stage A1. In that stage, the two posteriormost structures, the otic capsule and the parachordal, are the first structures which Orbital cartilage and trabecular horn differentiate into a precartilaginous Anlage at stage D4 (Fig. 3b) (Fig. 7).

Comparison of D. scovazzi, X. laevis, B. orientalis
We used data from earlier studies describing the chondrogenesis in X. laevis [21] and B. orientalis [25] to compare our recent results. We scored additional cartilaginous features in both species to build a uniform database for future comparison with a wider range of species. We scored 11 additional cartilaginous features for X. laevis and 22 for B. orientalis (Tables 2, 3). Some structures, like the admandibular cartilage and urobranchial process of the basibranchial are not present in X. laevis and B. orientalis and are therefore not suitable for a comparison with D. scovazzi. In X. laevis and B. orientalis the external morphology does not give many indications about the developmental sequence of chondrification of the cranial skeleton. However, in D. scovazzi there is a clear correlation between the external morphology and the progress of skeletogenesis; i.e., in specific Gosner stages specific chondrocranial features are present. Another peculiarity in D. scovazzi is the simultaneous transition of numerous structures from mesenchymal to prechondrogenic Anlage at stage D4 (Fig. 3a,b) and from prechondrogenic to chondrogenic Anlagen at stage C2 (Figs. 2, 3-6). In X. laevis and B. orientalis the differentiation is more stepwise and extended over several stages. Not only is it possible to conclude from the developmental stage to the state of cranial skeletogenesis in D. scovazzi, but the species also shows some sequential features not observed in the other species studied here. The mesenchymal Anlage of the infrarostral cartilage is among the last appearing Anlagen during the cranial skeletogenesis in D. scovazzi, while it is one of the earliest mesenchymal Anlagen of the cranial skeleton in X. laevis and B. orientalis. Additionally, only one infrarostral Anlage is present in D. scovazzi instead of two as observed in the other two species. The  While there are characters where D. scovazzi stands out, there are also several shared features of cartilaginous development with its close relative B. orientalis. Regarding the suprarostral cartilage, in both species the alae differentiate before the corpus of the suprarostral and the mesenchymal Anlage of the posterior process develops as the last element of the suprarostral cartilage. Additionally, the chondrification of the whole cartilage only takes two stages. Another commonality is that the ventromedial and retroarticular processes of Meckel's cartilage There were also shared features of all three compared species D. scovazzi, B. orientalis, and X. laevis; one is that the development of the palatoquadrate is relatively similar. The palatoquadrate develops early and no anterior to posterior developmental sequence was observed. However, several other structures develop in an anterior to posterior sequence. Such a sequence is present during the development of the neurocranium-anchoring structures of the palatoquadrate (quadratocranial commissure, ascending process, larval otic process). The larval otic process, which is not present in D. scovazzi, is the last part of the palatoquadrate that chondrifies in X. laevis and B. orientalis. Furthermore, an anterior to posterior differentiation pattern is also observable in the branchial basket of all three species, including the ceratobranchials I-IV as well as the proximal and terminal commissure I-III.

Additionally notes for Xenopus and Bombina
The sequences were updated from Lukas and Olsson [21,25] and the updated tables are presented here (Tables 2,  3).

Larval skeletal characters and their phylogenetic implications
Most of the skeletal features of Discoglossus scovazzi are congruent with descriptions made in D. pictus and D. sardus [27][28][29]. But some features are different in D. scovazzi. The hypobranchial plate is fused to ceratobranchial I in contrast to the synchondrotic articulation between hypobranchial I (the anterior part of the hypobranchial plate) and ceratobranchial I described in D. pictus [27]. The presence of the spicules I-III, a postcondylar process at the posterior margin of the ceratohyal and the presence of transversal tectal taenia were mentioned as defining features of the genus Discoglossus [28,29]. However, these structures are absent in the investigated stages of D. scovazzi (here studied up to Gosner [26] stage 29 = Go29). If the absence is an apomorphic trait of D. scovazzi or if these structures appear later during development (Haas [29] studied Go37 and Púgener [28] Go34 tadpoles) requires further investigation.
An admandibular cartilage is present in a similar condition as described in other species of Discoglossus [28,30], the closely related Alytes [29,30], and the basalmost neobatrachian Heleophryne [31][32][33]. Among Discoglossoids, species of Bombina lack this cartilage [25,29]. Therefore, the presence of the admandibular cartilage might be an apomorphic feature of the Alytidae (comprising Alytinae and Discoglossinae) and distinguish them from the Bombinatoridae, as also suggested by recent molecular based phylogenies [34][35][36]. Further features which separate both clades are the absence of the larval otic process of the palatoquadrate and the lack of a synchondrosis between the trabecular horns and the suprarostral cartilage in Alytids [28,30,37] whereas both are present in Bombinatorids [25]. An inconspicuous additional feature of Alytids is the consistent width of the trabecular horns [30] while the width of them is terminally expanded in Bombinatorids [25,37].
A common feature of all Discoglossoids investigated so far is the presence of two posterior processes of the ala of the suprarostral cartilage [21,25,29,30]. This feature qualifies as apomorphic trait for the Discoglossoidea. Infrarostrals which are not fused are present in Alytinae, Discoglossinae, Bombinatoridae, and the basal Ascaphidae [25,30,38]. Therefore, this unfused condition may be synapomorphic for all anurans. The suprarostral alae and the suprarostral corpus are separated in Ascaphus truei [39], whereas both are connected in the majority of the so-called archaeobatrachians. Therefore, even if a separated suprarostral appears frequently among neobatrachians, this may reflect the ancestral anuran state of this cartilaginous structure [37]. The presence of a hyoquadrate process in all basal anurans also qualifies as a synapomorphic trait [37].
X. laevis has a specialized filter feeding larvae and while Pipidae are Mesobatrachia, several of the abovementioned features cannot be found. Paired infrarostrals are present in ascaphids, discoglossoids, scaphiopodids, pelodytids and pelobatids [25,30,37,38,40,41]. Megophrys and Xenopus have fused infrarostrals [21,42], whereas Hymenochirus, Pseudhymenochirus, and Rhinophrynus have no infrarostrals at all or they are fused to Meckel´s cartilage [21,43,44]. X. laevis also differs from the cartilaginous development of B. orientalis and D. scovazzi because of the bipartite origin of the palatoquadrate. X. laevis possesses two mesenchymal Anlagen of the palatoquadrate. The anterior Anlage extends posteriorly during further development and gives rise to the muscular process, the quadratocranial commissure and the subocular bar. The posterior Anlage grows anteriorly and gives rise to the ventrolateral process and the ascending process [21]. If we consider the tentacular cartilage of X. laevis as elongated lateral outgrowth of the fused suprarostral alae, then X. laevis contradicts the Discoglossoid sequence where the suprarostral corpus develops after the suprarostral alae. Instead, the suprarostral corpus, which is part of the suprarostral plate, chondrifies before the suprarostral alae (tentacular cartilage) in X. laevis [21].

Developmental sequence
The viscerocranium of chondrichthyans [17,18], teleosts [14], and sturgeons [20] develops in strict anterior to posterior direction. We have shown that basal anurans differ from this ancient pattern [21,25]. In B. orientalis and X. laevis the ceratohyal, an element of the hyoid arch, is the first cartilage to chondrify during development while the infrarostral and suprarostral cartilages, elements of the mandibular arch, develop much later. We assumed that the early development is linked to the importance of the ceratohyal as the central element of the buccal pump which draws water into the buccal cavity and enables proper inspiration and expiration. Furthermore, we assumed that the early development of the ceratohyal may be an apomorphic trait for the Lalagobatrachia (all anurans except the basal Ascaphidae and Leiopelmatidae). Both assumptions are challenged by the sequence observed in D. scovazzi. The ceratohyal has an interesting developmental trajectory. The mesenchymal Anlage of the ceratohyal develops before the mesenchymal Anlage of the suprarostral ala and subocular bar, but simultaneously with the mesenchymal Anlage of the quadratocranial commissure, ceratobranchial I and hypobranchial plate. Chondroblasts appear earlier in the ceratohyal than in those five elements, but all those elements finish their chondrification before the ceratohyal. Furthermore, studies comparing muscle developmental sequences in vertebrates suggest that the mandibular arch-derived structures regularly appear simultaneously or after hyoid arch-derived structures [12], which would support the hypothesis proposed by Miyashita [13] that the mandibular arch only became secondarily integrated with the ancestral pharyngeal arches (hyoid and branchial arches) in the transition that lead to the last common ancestor of crown-group Gnathostomata and only became secondarily similar to them. With the current available developmental sequences it is not possible to verify the ancestral sequence in amphibians but further studies are underway.
There is also no strict anterior to posterior chondrification pattern within the viscerocranium of D. scovazzi. Hyoid arch elements (e.g., basihyal) chondrify after elements of the branchial arch (e.g., ceratobranchial I-IV) and mandibular arch elements (e.g., infrarostral cartilage) chondrify after elements of the branchial arch I (e.g., ceratobranchial I). Therefore, we can confirm that the development of the viscerocranium of anurans is a complex mosaic and does not follow a strict directional pattern. The ceratobranchials I-IV develop more simultaneously in D. scovazzi than in B. orientalis, X. laevis, Rana temporaria, Bufo cinereus, and Hyla sp. [21,25,45,46] where they develop in a stepwise anterior to posterior sequence. However, the chondrification of the neurocranium anchoring processes of the palatoquadrate (quadratocranial commissure and ascending process) follows a clear anteroposterior sequence in D. scovazzi as was described before in several other anuran species [21,25,45,46].
In Ascaphus truei the lateral parts of the suprarostral chondrify later than the medial parts [39]. In B.orientalis and in D. scovazzi the lateral alae develop before the medial corpus of the suprarostral cartilage [25]. The suprarostral is not present as a separate element in X. laevis as it is fused with trabecular horns, anterior process of ethmoid plate; the medial suprarostral plate develops before the lateral tentacular cartilages. This leads to four possible scenarios. The mandibular arch derived cartilages, Meckel's and palatoquadrate, originate from a single mesenchymal anlage, which is the ancestral gnathostome condition [17,47]. The mesenchymal anlage separates into the ventral anlage of the Meckel's cartilage and the lateral anlage of the palatoquadrate. The later chondrification of both cartilages is independent of each other. In anurans, two separate distinct mesenchymal Anlagen of Meckel's cartilage and palatoquadrate exist at the earliest investigated specimens [21,25,45]. However, since several developmental studies have shown that the continuous anlage of the first branchial arch will give rise to the upper and lower jaw elements as a response to genetic patterning [e.g., Dlx pattern, 48], it is reasonable to assume that this is the case in amphibians too.
Comparing the general appearance of branchial arch derived Anlagen and ignoring the individual processes of specific cartilages (Tables 1-3) then we can make following observations: In D. scovazzi the mandibular, hyoid, and first branchial arch Anlagen develop first followed by stepwise addition of the branchial arches II, III, and IV. In B. orientalis the mandibular and hyoid anlagen appear first, followed by anlagen of ceratobranchial I and II, and a stepwise addition of the other two ceratobranchials. In X. laevis mandibular, hyoid, and first and second branchial arch Anlagen develop first followed by third and fourth ceratobranchial Anlagen in the next step. This anteroposterior development of pharyngeal arches with simultaneous occurrence of mandibular and hyoid arch derivatives was also described for several amphibians, including B. orientalis and X. laevis [12,49], but in fishes the mandibular arch anlagen often appear after the hyoid arch Anlagen (Scyliorhinus canicula: [12]; Polypterus senegalus: [50]; Danio rerio: [51]; Neoceratodus forsteri [11]:). In aves and in Theria (marsupials and placental mammals) there seems to be some variability (Gallus domesticus: [9]; Coturnix coturnix: [52]; Monodelphis domestica: [53]; Mus musculus: [52]). The evolutionary significance of this appearing pattern is not obvious to us and might require the analyses of a wider range of taxa to evaluate for eventual functional or developmental constraints.
The neurocranium of several osteichthyans (bony fishes; e.g., Japanese medaka: [14], sturgeons: [20], coelacanth: [23], Australian lungfish: [55]), "reptiles" [16,19,22,23,56,57] and birds [24] develop in posterior to anterior direction. Otic capsules, parachordals, and cranial trabeculae are the first centers of chondrification and extend anteriorly to build the neurocranial elements in these species. Once again, anurans differ from this gnathostome pattern. Parachordals (C1) and otic capsules (C2) are among the first structures to chondrify in A. truei and D. scovazzi ([39], present work). These posterior parts of the neurocranium extend anteriorly, while the anterior parts of the neurocranium, the trabecular horns and the orbital cartilage extend posteriorly until both parts meet and fuse. Neurocranial structures develop later in X. laevis and B. orientalis, but the anterior growth of posterior elements and the posterior growth of anterior elements is also present [21,25]. The late development of the synotic tectum is not only a feature of anurans [21,25,39,45] but also present in avians and the Australian lungfish [24,55]. Additionally, the chondrification of the otic capsule from lateral to medial seems to be conserved among anurans [21,25,39,45].

Conclusions
We compared the timing of the cranial chondrification of three basal anuran species and hypothesize the following features of anuran cartilage formation: 1. The viscerocranial elements do not chondrify in the presumably ancestral anterior to posterior direction.
2. The neurocranial elements do not develop in the presumable ancestral posterior to anterior direction. Instead, the posterior elements extend anteriorly and the anterior elements extend posteriorly direction until both parts meet. 3. The neurocranium-anchoring processes of the palatoquadrate chondrify in anterior to posterior direction; first the quadratocranial commissure, then the ascending process, and last the larval otic process. 4. The infrarostral cartilage and the proximal commissure I chondrify simultaneously. 5. The palatoquadrate and basibranchial chondrify simultaneously. 6. Anterior to posterior developmental sequences can be observed best in the branchial apparatus development.
The here presented sequences in anurans support that the posterior to anterior direction of development and growth of the parachordal as well as the initial development of the lateral wall of the otic capsule are shared gnathostome features.

Husbandry and staging
Fertilized eggs of Discoglossus scovazzi were provided by a private breeder. Approximately 20 eggs per petri dish were cultured in 0.1X modified Barth's saline [58]. They were kept at temperatures between 18 and 25 °C to ensure that every stage of interest can be collected (3-5 tadpoles every 2 h). All specimens (embryos and larvae) were staged according to the simplified staging table for anuran embryos and larvae [26] and denominated as "Go stages. " A developmental series was established by collecting embryos and larvae from defined stages between Go 20 and Go 34 (n = 156). Anesthesia was performed using 1% tricaine methanesulfonate (MS-222) according to the animal welfare protocols at the Friedrich-Schiller-University Jena. Specimens were fixed in 4% phosphate-buffered formalin (PFA). Histological sections and cleared-and-stained larvae are kept at the Institute of Zoology and Evolutionary Research, Friedrich-Schiller-University, Jena, Germany.

Tissue staining
The PFA-fixed specimens were dehydrated, embedded in paraffin, and serial sectioned at 7 μm thickness using a rotary microtome (Microm, HM 355 S). The sections were stained according to Heidenhain's Azan technique [59]. Images were taken with an Hitachi HV-F202SCL camera mounted on an Zeiss AxioScan Z1 microscope operated with Zen 3.1 software. The clearing-and-staining procedure followed the protocol by Dingerkus and Uhler [60] with the exception that no alizarin red was used due to the absence of bones. Cleared-and-stained specimens were examined with a Zeiss Stemi 11 and images were taken by an attached camera (ColorView) operated by AnalySIS software.

Image processing
The digitized images (TIFF-format) of the respective histological sections were exported to Fiji Software [61]. The section images of each individual were stacked and aligned using first the least squares (rigid) and second the elastic non-linear block correspondence mode from the TrakEM2 plugin for Fiji [62]. Elastically aligned stacks were exported as tagged image files (TIFF). Segmentation of the different skeletal structures was performed in Amira 6.0.1. 3D analysis software (FEI Visualization Sciences Group). Polygonal surfaces were rendered and then exported to Wavefront OBJ file format for further processing in Autodesk Maya 2021 (Autodesk, Inc.). Surfaces were smoothed, polygonal counts reduced, and the surfaces arranged. For the final composition, coloring and rendering of images, Autodesk Mudbox 2021 (Autodesk, Inc.) was used. All images were edited and arranged using Adobe Photoshop CS6 and Adobe Illustrator CS6 (Adobe Inc.).

Scoring
We defined and scored a total of 75 cartilaginous structures (cartilages, processes, etc., incl. joints) in each investigated specimen of D. scovazzi (44 out of 75 are present). To enable a proper comparison, earlier descriptions from X. laevis and B. orientalis [21,25] were updated to enable maximal comparable structures. Terminology used here follows the guidelines introduced by Haas [29] for cartilaginous features (anglicized terms). The following different states of cartilaginous development were scored: (1) cartilaginous structures are absent; (2) mesenchymal Anlagen are visible as condensed cell clusters; (3) chondroblasts form condensed precartilaginous cell clusters with clearly visible nuclei. (4) chondrocytes rich in cytoplasm and bordered by a clearly visible perichondrium are present. All structures were scored after the first appearance principle. If a certain state was visible within the respective structure, the whole structure was scored in this state even if parts of the structure remained in an earlier state. Additionally, we defined different stages of development. Each stage is defined by a specific combination of cartilages in different cartilaginous developmental states. A1 (Anlage 1) starts with the appearance of the first mesenchymal Anlage, D1 (Differentiation 1) starts with the appearance of the first Anlage which contains chondroblasts and stage C1 (Cartilage 1) starts with the appearance of the first chondrocyte containing cartilage. The following enumeration of stages is the result of additional cartilaginous structures entering different states.